The organization of the nucleus into distinct structural and functional domains is important for normal chromosomal processes like transcription and replication and disruption of this organization is known to lead to disease states like cancer and developmental defects. Thus understanding how the nucleus is organized and responds to stimuli should help us better manipulate processes for benefit in research and medicine. The overall objective of this proposal is to reach a mechanistic understanding of the structure and organization of domains in eukaryotic chromosomes. Adjacent domains in the nucleus often have antagonistic structural or functional properties and determining the precise structural and molecular properties of different domains will be necessary to understand how the nucleus is organized. While DNA insulator elements have been identified that separate distinct functional domains, the elements that separate the structurally defined chromosomal domains are not well defined. Understanding the relationships between the functional and structural elements that organize the nucleus is a key focus this proposal. The mechanisms involved in separating distinct chromatin domains is just beginning to be understood and likely involve post-translational modifications of histones as well as attachment of chromatin loops to proteinaceous superstructures, many mechanistic questions remain and is also an area of inquiry proposed in this research grant. The research will involve use of molecular genetics and genomics, manipulation of chromatin using techniques like quantitative chromatin immunoprecipitations, and chromosome conformation capture and biochemical analysis to identify factors regulating insulator elements and chromatin loop formation. Given that many nuclear proteins and the basic structure of chromatin is conserved from yeast to humans, these studies will help discern general principles of chromatin domain dynamics that can be extrapolated to gene regulation in humans. Since alterations in chromatin architecture is known to cause states like cancer and developmental defects, the results from this study will be useful in developing new strategies for treating these disease states and also in developing better molecular tools for gene therapy.